HIV/AIDS is a major cause of global morbidity and mortality. Direct killing of infected CD4+ T cells and evasion of immune responses underlie the pathogenesis of AIDS. The membrane-associated viral Nef protein, which is critical for viral replication and virulence downregulates many immune cell receptors including CD4 and MHC class I. In the previous reporting period(s), we showed that both the recycling cell surface receptor and the nascent receptor in transit to the plasma membrane were susceptible to intra-cellular retention and degradation by HIV-1 Nef (J Cell Biology, 163, 1281, 2003 and J Biol Chem. 280, 7413, 2005). Genetic and biochemical studies have suggested two mutually exclusive mechanisms for HIV-1 Nef induced and HLA-I downmodulation: 1) a multi-step hierarchical dysregulation of PACS-1 and PI3K dependent ARF6 endocytic pathway;or 2) by AP-1 vesicle dependent rerouting of newly synthesized MHC-1 complex from the TGN to the endolysosomal pathway for degradation. We found that 1) Nef mediated loss of endogenous HLA-I at the plasma membrane of HeLa cells and PBMCs was reversible by genetic inhibitors of ARF6, Rab7 and Rab11 GTPases that regulate endocytic traffic. While siRNA knockdown of clathrin, AP1 adapter(s) reversed this Nef phenotype in HeLa cells and PBMCs, knockdown of PACS-I and to a less extent ARF6 and ARNO GEF was also effective in HeLa cells. In contrast, AP1 or clathrin knockdown was able to reverse Nef effect on endogenous HLA-I in Jurkat cells, while the genetic inhibitors were ineffective. 2) In contrast, the profound loss of cell surface expression of HLA-I A2 in Jurkat that was not rectified by genetic inhibitors of endocytosis and was reversed only by knockdown of AP1 adapter(s) or clathrin. The much less potent Nef effect on HLA-I A2 in HeLa cells was partially susceptible to genetic inhibitors of ARF6 and Rab GTPases and to genetic knockdown of AP1, clathrin or PACS-1. 3) Ikarugamycin, an inhibitor of clathrin pit mediated endocytosis, which was effective in blocking Nef mediated downregulation of CD4 in different cell types was only able to block Nef induces downregulation of endogenous HLA-I in HeLa cells and quiescent PBMCs. 4) The three cardinal mutations, M20A, E62A and P72A were more efficient in reversing the Nef effect on endogenous HLA-I in HeLa cells and PBMCs. In sum, our findings indicate, Nef subverts both antero- and retrograde trafficking MHC-I alleles, more predominantly the anterograde trafficking of coexpressed HLA-I A2 in T cells than in HeLa cells while impairing the endocytic traffic of endogenous HLA-I predominantly in HeLa cells and quiescent PBMCs. These conclusions are further supported by realtime analyses of interaction of Nef with HLA-I A2 or CD4 in various subcellular organelles by FRAP microscopy and FCS spectroscopy. Chemokine receptors constitute a sub-class in GPCR (the G-protein coupled receptors) superfamily, which is phylogenetically conserved from yeast to primates and regulate diverse biological processes besides leukocyte chemotaxis. In previous years a) we identified the structural requirements of chemokine receptors for the biological function and HIV usage (J Biol Chem. 276, 40133, 2001);b) investigated how naturally occurring CCR5 mutants impaired for surface expression impact the physiological function of the wt receptor and its HIV usage in heterozygotes (J Biol Chem. 277, 2287, 2002);c) addressed the mechanistic differences between the CCR5 and CXCR4 receptors in the agonist-driven receptor signaling, and trafficking (Mol Biol of Cell 14, 3305, 2003), d) showed that in primary leukocytes, agonist driven cell migration and receptor internalization are separable processes (J. Biol Chem. 279, 24372, 2003);and e) showed that cholesterol was absolutely required for chemotaxis and associated spatial polarization of chemotactic mediators, but not for endocytosis of agonist-occupied receptors and associated signaling (J Biomed Sci 15, 441, 2008). Human chemokine and chemokine receptor dysfunction are associated with numerous acute and chronic diseases such as asthma and arthritis and infectious diseases including AIDS and TB. Our understanding of this feedback controlled signaling system in health and disease will enable us to understand viral pathogenesis and help us develop novel therapeutic interventions. HIV-1, HIV-2, and SIV Nef alleles are known to downmodulate several chemokine receptors. We show that Nef downmodulates some chemokine receptors to a variable extent based on the cell type, receptor, and primary cell donors. Of all the chemokine receptors examined, CXCR4 in different primary cells and tumor cell lines and CXCR1 and CXCR2 in certain cell lines were downregulated by different HIV and SIV Nef alleles in the context DNA transfection, single-cycle HIV infection or Nef protein transduction, although the downregulation was quite modest compared to that of CD4. We also show that both naturally truncated mutants of CXCR4 as in WHIMS or engineered truncated variants of CXCR1, CXCR2 and CXCR4 (which lack classical motifs for endocytosis) are downregulated more profoundly than wt receptors. Nef induced ubiquitination of CXCR4 through recruitment of E3 ligase, AIP4 followed by a dynamin dependent endocytosis into MVBs for proteolyis. These mechanisms were confirmed by interaction between Nef, AIP4 and CXCR4 and by reversal of Nef effect through siRNA knockdown of AIP4 or by a small molecular weight inhibitor of dynamin. Nef has been shown to inhibit agonist driven chemotaxis through CXCR4 and CCR5 receptors by subverting chemotactic signaling at multiple steps. We show that although Nef induced only a slight reduction of CXCR4 at the cell surface, the residual receptor was profoundly impaired for chemotaxis and ligand mediated receptor internalization. The chemotactic defect resulted from inhibition of early signaling steps such as dissociation of G-protein subunits, intracellular Ca++ flux, polarized accumulation of pAKT, F-actin polymers and other signaling molecules. These defects were a direct result of a specific proteolysis of Gai2 GTPase subunit but not of other G&#945;i isoforms or family members. Loss of G&#945;i2 subunit was profound and was comparable to that of Nef induced CD4 degradation and was demonstrable in many cell types. Reconstitution with exogenous G&#945;i2 or other isoforms did not significantly rectify the chemotaxis defect. Unlike Nef induced loss, siRNA mediated G&#945;i2 KD did not diminish CXCL12 dependent signaling or alter CXCR4 expression. By degrading one specific Gai subunit, Nef might induce leukocyte migratory and homing defects in AIDS pathology. The members of the Toll-like Receptor (TLR) family play an important role in innate immunity. Of the 11 TLRs that have been identified, TLR1/TLR6, TLR2 and TLR4 form homo- or hetero-dimers, which can recognize various bacterial lipopeptides. These TLRs on the cell surface of leukocytes regulate chemotaxis, phagocytosis, and immune activation. TLR ligand LPS at 0.02 ng/ml over 15 min inhibited chemotactic response of CCR2, CXCR4 and FPR in monocytes, but not in PMNs, T cells or myelomonocytic cell lines. LPS treatment abolished polarized accumulation of F-actin and PIP3 in response to chemokine stimulation in monocytes but not in PMNs. TLR stimulation did not alter initial G-protein dependent signaling or the kinetics of endocytosis of occupied chemoattractant receptors. Chemotaxis inhibition following short-term LPS treatment didnt result from downmodulation of signaling receptors or from inordinate increase in adhesion through integrin activation. However, chemokine stimulation of LPS treated monocytes displayed circumferential accumulation of activated integrin and F-actin polymers resulting from global recruitment of EPAC leading to unpolarized activation of Rap1.